Ready-To-Go technology for convenient use of biomolecules is based on a mixture of excipients combined with lyophilisation to provide long-term room temperature stability to biomolecules and reagents including proteins, enzymes etc. The stability is attributed to the fact that the lyophilized biomolecules or proteins exist in an amorphous glassy state that markedly reduces molecular mobility and subsequent reactivity. The glass transition temperature (Tg) for a lyophilized product indicates the temperature at which the glass state begins to transition to a more mobile flexible state, allowing molecular mobility and biomolecule activity to return but with a consequential loss of stability. Knowledge of the Tg value is thus important, as a lyophilized product must be stored below this temperature for long-term stability. Ready-To-Go having Tg values well above room temperature ensure that the products can be shipped and stored without the need for refrigeration. Since the introduction of the first Ready-To-Go product, the technology has demonstrated numerous benefits for the stabilization of a variety of reagents and complex assay mixtures. The technology is suitable for use with sensitive low stability enzymes, antibodies, primers, probes, dyes and other reagents. The physically stable, solid lyophilised reagent provides long term stability at ambient temperature, can be dispensed in flexible formats, and is amenable to downstream manipulation. The compatibility of Ready-To-Go technology with complex mixtures enables the formulation and stabilization of pre-dispensed, single-dose reagents and reaction mixtures. This format can reduce cross-contamination risk and improve data reliability by requiring fewer pipetting steps and less handling overall. Fewer assay steps supports reduced training requirements and simplifies automation of the process.
The temperature stability of Ready-To-Go stabilized materials simplifies and reduces costs for shipping and storage. These materials do not require dry- or wet-ice shipment, which simplifies shipping across countries. Items can be shipped to remote regions or regions with insufficient infrastructure without the concerns or costs associated with temperature-controlled shipments. Once the materials arrive, they can be stored at ambient temperature at the point of use, in the field or lab. Preferably Ready-To-Go products and reagents are heat-sealed under low humidity conditions and stored at ambient temperature in an airtight foil pouch with desiccant.
Taq DNA polymerase Ready-To-Go PCR Beads (GE Healthcare) consist of a pre-formulated, pre-dispensed, freeze-dried PCR reagents mix for single-dose reactions delivering robust and reproducible performance in standard PCR amplifications. The beads and the constituent reagents are room temperature stable and as such offer significant advantages over traditional PCR amplification workflows. A bead is defined as a spherical shape.
The long-term ambient-temperature stable property means that no specialised freezer space is required and therefore less energy is consumed when shipping and storing the product. When being used researchers simply add the template DNA solution and primers of interest and initiate the appropriate thermo-cycle. The bead format reduces the need for multiple pipetting steps and as such minimises the risk of pipetting errors and DNA contamination thereby delivering a system that is associated with more reproducible results. The beads consist of high-quality recombinant Taq DNA polymerase and high-purity reagents. All of these facilitate the delivery of a highly robust and reproducible performance in the majority of downstream applications such as PCR. The use of recombinant Taq DNA polymerase and other high-purity reagents ensures reliable and robust performance in both end point and real-time fluorescence-based PCR amplifications, and ensures the lowest possible levels of contaminating prokaryotic and eukaryotic nucleic acids. An additional advantage is the beads have been verified for use in real-time quantitative PCR workflows.
The Taq DNA polymerase Ready-To-Go PCR Beads (GE Healthcare) are pre-formulated to ensure greater reproducibility between reactions, minimize pipetting steps, and reduce the potential for pipetting errors. The only additional reagents required are water, primers, and template DNA. The beads are provided pre-dispensed into either 0.2 ml or 0.5 ml PCR tubes. The 0.2 ml tubes are also supplied in a 96-well (8×12) plate format that allows individual strips of eight tubes to be easily removed. This flexibility allows use of the entire 96-well plate, strips of eight, or individual 0.2 ml tubes. When reconstituted, each bead contains ˜3.5 units of recombinant Taq DNA polymerase, dATP, dCTP, dGTP, dTTP, stabilizers, BSA and reaction buffer. When a bead is reconstituted to a 25 μl final volume, the concentration of each dNTP is 200 μM in 10 mM Tris-HCl (pH 8.0 at room temperature), 50 mM KCl and 1.5 mM MgCl2.
US 2009/0325263 describes Ready-To-Go PCR cakes (Preparation of glassified biological reagents) formulated in a similar manner to Taq DNA polymerase Ready-To-Go PCR Beads. A cake is defined as non-spherical in shape e.g. concial, flat, square etc, the actual shape is determined by the shape of the receptacle into which the mix is dispensed into.
Current Ready-To-Go beads and cakes consist of the anionic detergent Rodafac RE-960 as a means to stabilise the reagents for ambient temperature storage and use at elevated temperatures in procedures such as PCR, which is described in US 20100159528 A1.
A drawback with current Ready-To-Go beads and cakes is that there appears to be a significant amount of electrostatic energy associated with beads and cakes. This is a particular disadvantage especially for beads dispensed into receptacles such as PCR tubes, Eppendorf centrifuge tube, 96-well PCR and flat-bottomed plates etc as the tendency for the beads to “jump” out of the receptacle due to either electrostatic attractive or repulsive forces. It would be desirable to have an improved manufacturing process providing bead/cakes which exhibit reduced attractive and repulsive forces.